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Microbial synthesis of aromatic (plant) natural products and other aromatic molecules of biotechnological interest

Natural products are metabolites of the secondary metabolism that are not directly involved in growth, development or reproduction, but have important functions in defense and signaling, or serve as pigments or fragrances. Of the many hundreds of thousands natural products known to date, many demonstrate important pharmacological activities or are of biotechnological significance. However, isolation from natural sources is usually limited by low abundance in the producing organisms, whereas total chemical synthesis is typically commercially unfeasible considering the complex structures of most natural products. With advances in DNA sequencing and recombinant DNA technology, many of the biosynthetic pathways responsible for the production of these valuable compounds have been elucidated, offering the opportunity of a functional integration of biosynthetic pathways originating from plants or other microorganisms in the production strain of choice [1,2].

We apply latest molecular tools in the high-throughput format for the construction of tailor-made recombinant Corynebacterium glutamicum strains by metabolic engineering to modify metabolic profiles according to individual production purposes. This includes the heterologous expression of metabolic pathways to confer the capability for natural product synthesis, but also host cell engineering for optimized substrate utilization, improved precursor supply, reduced product degradation or product secretion [3]. This approach offers the promise to provide sufficient quantities of the desired natural products from inexpensive and renewable resources [4,5].

Graphic Overview of the aromatic compounds of biotechnological interest for whose synthesis Corynebacterium glutamicum strains have been developed by the Synthetic Cell Factories Group



References:

[1] Milke L. and Marienhagen J. (2020). Engineering intracellular malonyl-CoA availability in microbial hosts and its impact on polyketide and fatty acid synthesis. Appl. Microbiol. Biotechnol. (epub ahead of print). (https://doi.org/10.1007/s00253-020-10643-7)

[2] Kallscheuer N., Classen T., Drepper T., Marienhagen J. (2019). Production of plant metabolites with applications in the food industry using engineered microorganisms. Curr. Opin. Biotechnol. 56: 7–17. (https://doi.org/10.1016/j.copbio.2018.07.008)

[3] Milke L., Kallscheuer N., Kappelmann J., Marienhagen J. (2019). Tailoring Corynebacterium glutamicum towards increased malonyl-CoA availability for efficient synthesis of the plant pentaketide noreugenin. Microb. Cell Fact. 18: 71. (https://doi.org/10.1186/s12934-019-1117-x)

[4] Milke L., Mutz M., Marienhagen J. (2020). Synthesis of the character impact compound raspberry ketone and additional flavoring phenylbutanoids of biotechnological interest with Corynebacterium glutamicum. Microb. Cell Fact. 19: 92. (https://doi.org/10.1186/s12934-020-01351-y)

[5] Kallscheuer N., Kage H., Milke L., Nett M., Marienhagen J. (2019). Microbial synthesis of the type I polyketide 6-methylsalicylate with Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 103: 9619–9631. (https://doi.org/10.1007/s00253-019-10121-9)

 

Substrates from Biomass for a Sustainable Industrial Biotechnology

Biomass-derived D-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. Corynebacterium glutamicum has already been engineered to grow with this pentose as sole carbon and energy source. However, all currently described C. glutamicum strains utilize D-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO2, in particular, when aiming for the synthesis of α-ketoglutarate and its derivatives (e.g. l-glutamate). Driven by the motivation to engineer a more carbon-efficient C. glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. glutamicum [1]. This five-step pathway enabled a recombinant C. glutamicum strain to utilize D-xylose in D-xylose/D-glucose mixtures and thus represents a good starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on D-xylose containing substrates. Optimization of D-xylose uptake and discovery of hitherto unknown endogenous enzyme (side-) activities in C. glutamicum allowed us to develop a strain variant, which requires the expression of a single heterologous gene encoding a dehydratase for establishing D-xylose utilization via the Weimberg pathway for growth and product formation [2-4].

Graphic Engineering of Corynebacterium glutamicum towards d-xylose utilization via the Weimberg pathway




References:

[1] Radek A., Krumbach K., Gätgens J., Wendisch V. F., Wiechert W., Bott M., Noack S., Marienhagen J. (2014). Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates. J. Biotechnol. 192: 156–160.
http://dx.doi.org/10.1016/j.jbiotec.2014.09.026

[2] Brüsseler C., Radek A., Tenhaef N., Krumbach K., Noack S., Marienhagen J. (2018). The myo-inositol/proton symporter IolT1 contributes to d-xylose uptake in Corynebacterium glutamicum. Bioresour. Technol. 249: 953-961. (http://dx.doi.org/10.1016/j.biortech.2017.10.098)          

[3] Tenhaef N., Brüsseler C., Radek A., Hilmes R., Unrean P., Marienhagen J., Noack S. (2018). Production of d-xylonic acid using a non-recombinant Corynebacterium glutamicum strain. Bioresour. Technol. 268: 332-339. (https://doi.org/10.1016/j.biortech.2018.07.127)

[4] Brüsseler C., Späth A., Sokolowsky S., Marienhagen J. (2019). Alone at last! – Heterologous expression of a single gene is sufficient for establishing the five-step Weimberg pathway in Corynebacterium glutamicum. Metab. Eng. Comm. 9: e00090. (https://doi.org/10.1016/j.mec.2019.e00090)

 

 

Development of Transcriptional Biosensors and Their Utilization for High-throughput Screening of Microorganisms using FACS

Metabolic engineering for the microbial overproduction of high-value small molecules is a highly complex process, which is dictated by a large number of parameters. The biosynthetic pathways leading to these products comprise multiple native or heterologous catalytic steps, each one representing a potential bottleneck when directing carbon flux toward target small-molecule production. Furthermore, the host organism’s native genetic regulation network and the interaction with the target pathway can impact final product yields.

Graphic Typical design of a transcriptional biosensor for the detection of small molecules in single cells




Efforts in the field of metabolic engineering are currently defined by the implementation of a series of rational design-based strategies to modify the host genome and pathway enzymes to achieve moderate product titers. To generate microbial production strains with higher productivities, we want to follow the long trail of successes in protein engineering and develop approaches to high-throughput screening of single cells. For this purpose, we design, construct and apply transcriptional regulators sensing molecules of biotechnological interest, which, in response to this signal, drive transcription of a fluorescent protein [1].

In combination with fluorescence-activated cell sorting (FACS), these biosensors enable the rapid screening of large libraries at the single cell level [2].

Graphic Biosensor-based ultra-high-throughput screening (uHTS) workflow using fluorescence-activated cell sorting (FACS) to isolate producing single cells from genetically diverse libraries


References:

[1] Eggeling L., Bott M., Marienhagen J. (2015). Novel screening methods - biosensors. Curr. Opin. Biotechnol. 35: 30-36. (http://dx.doi.org/10.1016/j.copbio.2014.12.021)

[2] Flachbart L. K., Sokolowsky S., Marienhagen J. (2019). Displaced by deceivers - Prevention of biosensor cross-talk is pivotal for successful biosensor-based high-throughput screening campaigns. ACS Syn. Biol. 8 (8): 1847-1857. (https://doi.org/10.1021/acssynbio.9b00149)